Genetic manipulation of many bacterial genera can often be difficult due to host restriction systems. These systems are designed to prevent foreign DNA from stably expressing and maintaining genes (e.g., bacteriophage) that may be disadvantageous to the host. As a result, very few, if any, transformants are obtained except in those rare cases where the process methods have been highly optimized. Even highly optimized methods can produce few transformants and often the results are irreproducible due to variations in physiological parameters of the host cells.
The genus Clostridia which include species of medical and industrial importance have historically been difficult to manipulate genetically. Those that have been genetically transformed are few and the efficiencies have been low, typically limited to at most 1×103transformants/μg DNA in the most efficient systems (Mermelstein et al., 1993, Applied and Environmental Microbiology, 59:1077-1081; Allen and Blaschek, 1990, FEMS Microbiology Letters, 58:217) but more often only yield a few recombinant colony forming units (CFU). Integrative plasmids have been used with even less efficiency, typically attaining transformation frequencies no higher than 1 CFU/μg DNA. The prevailing view is that the low transformation frequencies are due to the introduced DNA being degraded during the transformation process by one or more host restriction systems. In those cases where suitable transformation efficiencies in Clostridium have been observed and shown to be reproducible, it was often due to blocking of a specific endonuclease site encoding a restriction enzyme (e.g., CacI, C. acetobutylicum).
Thus, there remains a need in the art to improve the efficiency of introducing DNA into Clostridia. 